The primary structure of a mammalian NGF (mouse NGF) was first elucidated by Angeletti and Bradshaw, Proc. Natl. Acad. Aci. USA 68:2417 (1971). The primary structure of its precursor, pre-pro-NGF, has been deduced from the nucleotide sequence of the mouse NGF cDNA (Scott et.al. Nature 302:538 (1983); Ullrich et.al. Nature 303:821 (1983)).
The highly homologous human NGF (hNGF) gene has also been identified (Ullrich, Symp. on Quan. Biol., Cold Sprinz Harbor 48:435 (1983). Its homology to the mouse NGF is 90% and 87%, on the amino acid and nucleotide sequence levels, respectively. Due to the scarcity of hNGF, little is known about its biologic activity.
Baculovirus expression vectors have been used for the expression of a variety of foreign genes in insect cells either or in vivo (See, U.S. Pat. No. 4,745,051, issued May 17, 1988). These expression vectors enlist the highly efficient and temporally regulated Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin promoter with the gene of interest inserted downstream from the promoter. The corresponding gene product is obtained from Spodoptera frugiperda cells infected with the recombinant baculovirus vectors.
The isolation of the .beta.-subunit of hNGF and its expression as a heterologous protein in E. coli is described in European Patent Application 0,121,338. By using recombinant techniques, human .beta.-NGF was expressed essentially free from other mammalian proteins. Expression of the hNGF in E. coli using two genes which contain altered amino-termini resulting in the expression of a fused protein is described by Iwai, et al., Chem. Pharm. Bull. 34:4724 (1986)
The use of recombinant techniques to produce polypeptides in virally infected insect cells is described in European Patent Application 0,228,036. Using the baculovirus expression system, a method for the production of the protein, hepatitis B surface antigen, is set forth. The use of mixed polyhedral inclusion bodies (PIB) which contain a mixture of nucleocapsids from at least two genetically distinct baculoviruses, one of which contains the heterologous gene of interest, is described in Patent Cooperation Treaty (PCT) application WO 88/02030. Using the described coinfection technique a heterologous protein is expressed.
Although expression of these large mammalian proteins is possible, until the present invention there was little information on the successful expression of small molecules, such as hNGF, using a baculovirus expression system. It has also been questionable whether this particular expression system would be suitable for producing small proteins which require post-translational proteolytic cleavage of the protein precursor in order to exhibit biological activity.
We have now unexpectedly found that hNGF may be successfully expressed using a baculovirus insect cell expression system in good yields.
The instant invention describes the construction of expression vectors of use in a baculovirus system capable of expressing hNGF in a biologically active form. Production of sufficient amounts of hNGF by recombinant DNA technology according to this invention makes it possible to ascertain the potential utility of NGF in the treatment of Alzheimer's Disease (AD).